【作者】 陈德欣；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 植物学， 2007， 硕士

【摘要】 类胡萝卜素是人类饮食结构中不可缺少的重要成分，是植物中重要的色素之一，具有多种生物功能，在保护人类健康方面起着重要的作用。尤其是有些类胡萝卜素，如β-胡萝卜素是维生素A的前体，是人体维生素A的主要来源，具有强抗氧化作用，能够增强人体免疫力和具有防癌抗癌的功效，降低感染性疾病的发病率和死亡率，还能维持正常生殖能力。然而，人体自身无法合成β-胡萝卜素。必须通过外界摄入；但β-胡萝素在许多植物中含量较低，并且很难用化学方法合成。随着β-胡萝卜素生物合成途径的阐明及其相关基因的克隆，运用基因工程手段调控类胡萝卜素的生物合成已成为可能。因此，通过科学技术开发富含β-胡萝卜素的食品越来越受人们的重视。红肉蜜柚是珀溪蜜柚的一个新品种，其果实富含β-胡萝卜素，营养成分具比普通蜜柚更加丰富。本试验以红肉蜜柚为材料，进行β-胡萝卜素合成途径相关基因的克隆研究，以期为研究红肉蜜柚和其它果蔬β-胡萝卜素生物合成途径打下基础。主要试验结果如下：(1)建立了较为完善的样品红肉蜜柚总RNA提取和纯化方法。通过比较Trizol、申能博彩试剂盒、CTAB、改良CTAB四种RNA提取方法，总结出适应红肉蜜柚样品RNA提取的方法——改良CTAB法。并通过此方法获得了较为纯净的、完整的总RNA，可以直接用于后续的RT-PCR和RACE反应。(2)采用PCR扩增获得β-胡萝卜素合成途径系列酶基因的保守序列。以红肉蜜柚果肉总RNA逆转录的cDNA为模板，用扩增同源序列的简并引物，PCR扩增出PSY、PDS、ZDS 3个系列酶基因的保守序列，长度分别为662bp、599bp、569bp。(3)采用RACE方法获得PSY cDNA片段序列长1170bp，编码238个氨基酸残基，其推导的氨基酸序列与橙、温州蜜柑、葡萄柚、番木瓜、胡萝卜氨基酸序列相似性达98％、98％、97％、85％、85％。(4)采用RACE方法获得PDS cDNA片段序列长1612bp，编码468个氨基酸残基。其推导的氨基酸序列与橙、温州蜜柑、葡萄柚、番木瓜、胡萝卜等植物完整氨基酸序列的相似性分别为99％、98％、99％、90％、87％。更多还原

【Abstract】 Carotenoid is one of the most important classes of plant pigments,have a lot of diverse functions. They are importantly nutritional in human diets, especiallyβ-carotenoid has pro-vitamin A activity, it serves as precursors for vitamin A in our body.β-carotenoid plays an important role in human health. For example,β-carotenoid functions as antioxidant to improve the immune system positively, and also have the function of reducing the risk of suffering cancers or infectious diseases.However, human body can not synthesizeβ-carotenoid by itself,most ofβ-carotenoid are come from foods.But in many plants,they don’t have highβ-carotenoid contain, and it’s hard to synthesizeβ-carotenoid with chemical method.Therefore, people pay more attention to search or exploit the fruits with complex source of carotenoids using modern sciences and technologies.The red-fleshed grandis is a new breed of pummelo,it’s rich in nutrition, containing more vitamins andβ-carotenoid than general grandis. The red-fleshed grandis was used as testing materials, and the genes forβ-carotene biosynthetic were cloned to conduct relevant studies. Major results are descirbed as follow:(1) An improved method for RNA extraction from grandis samples was developed. In order to extract high quality RNA from grandis materials, four methods were compared, which included Trizol、CTAB、modified CTABand the reagent box, and the modified CTAB was the best one. Based on the method of modified Trizol, we obtained the pure and clean total RNA that could be used for further studies in the field of molecular biology such as PCR and RACE.(2) The consensus sequences of three genes ofβ-carotene biosynthetic pathway (psy, pds, zds) were cloned by PCR from the cDNA Reverse-transcripted from the RNA. And they were 422bp, 599bp and 569bp in length, respectively. (3) The RACE method was emplyed to obtain the 3’cDNA sequence of. The partial sequence of psy, which had a length of 1170bp and encodes a protein of 238 amino acids. Sequence analysis showed that its deduced amino acid sequence shared 98%、98%、97%、85% and 85% identity with that of psy of Citrus sinensis、Citrus unshiu、Citrus x paradise、Carica papaya、Daucus carota.(4) The RACE method was also used to acquire the 3’cDNA sequence of pds. The partial sequence of pds, which was linked by the 3’sequence and its consensus sequences, had a length of 1612bp and encodes a protein of 468 amino acids. Sequence analysis showed that its deduced amino acid sequence shared 99%、98%、99%、90%、and 87% identity with that of pds of Citrus sinensis、Citrus unshiu、Citrus x paradise、Carica papaya、Daucus carota.更多还原